The aims of this project are to assess the effects of aging and caloric restriction (CR) at a cellular and biochemical level of analysis, to identify physiological mechanisms associated with these effects, and to evaluate interventions/molecular pathways that might alter age-related declines in function. Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CR's beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. CR induces measurable changes on circulating levels of several hormones and growth factors that regulate cell growth and proliferation. Serum obtained from CR animals alters growth, proliferation and stress responses of cells in culture. We have demonstrated that it is possible to investigate certain aspects of CR using this in vitro approach. This approach lends itself to a more rapid investigation of possible mechanisms and, perhaps more importantly to the research, development and rapid evaluation of interventions that would be able to induce or promote a phenotype similar to that seen with CR, essentially a CR mimetic. Below is the description of two of the most prominent lines of work in my laboratory associated with this project. Mitochondrial Biogenesis and Caloric Restriction. CR is hypothesized to decrease mitochondrial electron flow and proton leaks to attenuate damage due to reactive oxygen species (ROS). We have focused our research on a related, but different anti-aging mechanism of CR. Specifically, using both in vivo and in vitro analyses, we reported that CR reduces oxidative stress by stimulating the proliferation of mitochondria through a PGC1a signaling pathway. These mitochondria under CR conditions show less oxygen consumption, reduced membrane potential and generate less ROS than controls but remarkably are able to maintain their critical ATP production. Thus, CR can induce a PGC1a-dependent increase in mitochondria capable of efficient and balanced bioenergetics to reduce oxidative stress and attenuate age-dependent endogenous oxidative damage. Carcinogenesis and Caloric Restriction. Almost a century ago Moreschi and Rous published their separate observations on the impact of caloric restriction (CR) on transplanted and induced tumors. Years later, McCay and colleagues first observed lifespan extension in laboratory rats maintained on a CR diet. Since then, CR has been studied intensively with consistent results showing its beneficial effects on longevity, age-associated diseases, attenuation of functional declines, and carcinogenesis across a variety of species and diet formulations. However, the mechanism(s) underlying the effects of CR protection still remain unknown. Nevertheless, it is safe to say that the three most extensively studied hallmarks of CR are enhanced protection against induced and spontaneous carcinogenesis, reduced insulin/IGF-1 signaling, and increased median and maximum lifespan. Even if CR was shown to benefit human health, confer cancer protection, and increase longevity, it would be extremely difficult to achieve adherence to such a stringent diet that might require a reduction of 20-40% in caloric intake. To this end, considerable investment has been focused on dissecting the pathways that regulate CR benefits that could spur development of pharmacological agents potentially acting as CR mimetics. Several of the currently proposed CR mimetics are phytochemicals (resveratrol, quercetin, and curcumin) that act, at least in part, through the activation of the NF-E2-related factor 2 (Nrf2) pathway. Nrf2 is a transcription factor that binds to the antioxidant response element (ARE) of target genes as an adaptive response to oxidative stress and increases the transcription of a variety of anti-oxidative and carcinogen detoxification enzymes. Stress can result from a variety of causes including fasting, overfeeding, endogenous compounds, exposure to chemicals or environmental agents but generally leads to the production of ROS. As a result of ROS exposure, Nrf2, which is typically bound to Keap1 in the cytoplasm, where it undergoes proteolytic degradation and rapid turnover, is phosphorylated and translocates to the nucleus where it binds to ARE sequences to induce expression of multiple cytoprotective enzymes including NAD(P)H-quinone oxidoreductase 1 (NQO1), glutathione S-transferases (GSTs), and heme oxygenase-1. We have now shown that Nrf2 is responsible for the protection of CR against carcinogenesis. However, the lack of Nrf2 did not attenuate lifespan extension or alter the CR improvement on insulin sensitivity in the Nrf2 KO mice. Similar to our findings with induced carcinogenesis, Van Remmen et al. were the first to show that reduction of an antioxidant enzyme could markedly increase DNA damage and spontaneous cancer incidence without affecting survival and lifespan. However, this study is the first to demonstrate that distinct pathways exert beneficial effects of CR and suggests that many mechanisms are involved in its protection. Recent data from invertebrates suggested that Nrf2 or at least some of its downstream effectors could hold the key to caloric restriction and longevity. But, it appears now in mammals that besides the involvement of Nrf2 on anti-carcinogenic protection by CR, there must be other pathways or factors that are involved in the regulation of mammalian longevity. Our data suggest that the lifespan extension seen in CR animals is not because of upregulation of the Nrf2 pathway and tumor response as suggested by the induced tumor studies. Finally and most importantly, the activation of the Nrf2 pathway is a promising target to evaluate for CR mimetics in the search for preventive strategies against environmentally induced cancers.